小鼠配对免疫球蛋白样受体及其介导的T细胞阶联与树突状细胞免疫耐受作用关系的研究
发布时间：2018-07-17 03:00
【摘要】： 移植物抗宿主病(GVHD)仍然是临床骨髓移植治愈血液系统恶性疾病的主要障碍之一。供者异基因淋巴细胞的异常活化是GVHD发生、发展的重要因素,诱导受者产生针对供者异基因主要组织相容性抗原(MHC)特异性耐受是预防GVHD、保存GVL的最佳途径。配对免疫球蛋白样受体(Paired immunoglobin-like receptor,PIR)是近年发现的主要表达在小鼠树突状细胞(DCs)上的免疫抑制性调节受体,包括免疫抑制性受体(PIR-B)及免疫活化性受体(PIR-A),其配体均是MHC-Ⅰ,二者与其配体的结合的水平是决定DCs免疫活化程度的重要分子标志之一。由于DC不仅是调控体内T细胞活化的关键,也是诱导免疫耐受的理想的靶细胞,可以直接或间接通过诱导供者抗原特异性调节性T细胞的生成、在体内形成T细胞阶联效应而抑制异基因T淋巴细胞的活化,因此研究PIR在树突状细胞的表达、T细胞阶联的关系及与免疫耐受的关系,可能为诱导供者MHC特异性耐受,实现GVHD及GVL的分离提供新的途径。 第一部分免疫球蛋白样受体在小鼠树突状细胞上的表达及与免疫耐受关系的研究 目的配对的免疫球蛋白样受体A、B(paired immunoglobulin-like receptor A、B, PIR-A、PIR-B)属于小鼠免疫球蛋白超家族成员。研究配对免疫球蛋白样受体PIR-A、B在小鼠DCs上的表达及与表面共刺激分子变化的关系,探讨PIR与免疫耐受的关系,探索诱导耐受性DCs的有效途径。 方法以C57BL/6小鼠来源DC系DC2.4细胞为研究对象,分别以重组小鼠白介素-10(recombinant mouse interleukin-10,rmIL-10)、重组人转化生长因子β1 (recombinant human transforming growth factorβ1,rhTGF -β1)诱导DC2.4细胞为耐受性DC(tolerogenic DC,T-DC),脂多糖(LPS)刺激48h为成熟DC2.4细胞(LPS-DC),体外化学合成特异PIR-B小RNA干扰片段(small interfering RNA,siRNA),以Lip2000转染DC2.4细胞(Si-DC)。分别应用半定量RT-PCR、流式细胞仪(Flow cytometry, FCM)及Western blot检测IL-10、TGF-β1、LPS及小干扰RNA对DC2.4细胞上PIR-A/B表达的影响;检测LPS刺激Si-DCs后表面共刺激分子CD80、CD86、MHC-Ⅱ及PIR-A的变化;以2-△△Ct表示各目的基因cDNA转录的相对表达量。分别以上述各组DCs细胞为刺激细胞,以异基因BALB/c小鼠脾淋巴细胞为反应细胞,应用3H-TdR标记法检测同种异体淋巴细胞的增殖能力(MLR),ELISA方法测MLR上清中IFN-γ的分泌水平变化。 结果FCM检测DC2.4细胞上PIR-A、PIR-B的共同的胞外区PIR表达的阳性率为(28.65±8.12)%,IL-10、TGF-β1及LPS诱导后PIR表达均上调(P0.05),分别为(54.21±6.34)%,(58.78±4.70)%,(48.24±6.75)%,但IL-10、TGF-β1及LPS各组间无显著性差别(P0.05)。半定量RT-PCR及Western blot显示,IL-10、TGF-β1诱导DC2.4细胞后PIR-B的mRNA及蛋白表达升高,而PIR-A表达则降低,而LPS刺激时则相反,PIR-A的mRNA及蛋白表达升高、PIR-B的表达则降低。流式细胞仪检测SiRNA阳性对照组的转染率为93.12%,SYBR greenⅠRealtime-PCR检测,LPS刺激后Si-DCs CD80、CD86、MHC-Ⅱ及PIR-A的表达高于正常DCs组。LPS-DCs组CD80、CD86、MHC-Ⅱ及PIR-A的2-△△Ct分别为5.02±1.09、4.69±1.75、5.46±1.79、6.02±2.13;LPS刺激后Si-DC组TAI分别为8.79±2.2、11.03±1.96、10.26±2.55、12.10±2.83,同LPS刺激的正常组相比,Si-DC组分别增加了3.72、6.34、4.8、6.08倍(P0.05)。混合淋巴细胞反应显示:正常DC2.4细胞可刺激异基因淋巴细胞反应,IL-10、TGFβ1诱导的T-DC组MLR明显受抑(P0.05),MLR上清中IFN-γ水平也相应降低(P0.05)。LPS-DC及Si-DCs组MLR明显增强(P0.05),MLR上清中IFN-γ水平明显增高(P0.05); 结论上调免疫抑制性受体PIR-B、下调活化性受体PIR-A是小鼠DCs获得耐受的普遍表型特征及分子生物学机制,沉默PIR-B的表达可使PIR-A及CD80、CD86、MHC--Ⅱ及PIR-A过表达,使DCs活化的机制,PIR-A和PIR-B构成了小鼠树突状细胞耐受的新靶点。 第二部分高度表达免疫球蛋白样受体B耐受性树突状细胞可诱导CD4+CD25+调节性T细胞生成 目的研究配对免疫球蛋白样受体B在树突状细胞上表达与调节性T细胞的生成的关系,探讨耐受性DCs诱导耐受的详细机制,为体内诱导耐受性DCs及调节性T细胞(Treg)生成提供实验依据。 方法免疫磁珠分选BALB/c小鼠CD4+T脾淋巴细胞。以rmIL-10(50ng/ml)、rhTGF-β1(50ng/ml)联合诱导C57BL/6小鼠来源的DC2.4细胞3天生成耐受性DCs(T-DC),同时设正常DC2.4细胞(DC)及LPS刺激48h后成熟的DC2.4细胞(mDC)干扰PIR-B组(Si-DCs)为对照组,各组DCs分别与BALB/c小鼠CD4+T脾淋巴细胞混合培养48h,检测Treg生成。RT-PCR检测转录因子Foxp3mRNA的表达变化,流式细胞仪检测CD4+CD25+Treg细胞的比例,PI检测CD4+T细胞的凋亡。磁珠分选的CD4+CD25+Treg与CD4+T细胞按照不同的比例加入MLR体系中,3H检测Treg对异基因DCs刺激的同基因淋巴细胞的增殖能力影响。 结果磁珠分选BALB/c小鼠脾CD4+T及CD4+CD25+T淋巴细胞纯度95%,正常DCs、T-DCs、Si-DC及mDCs各组细胞同BALB/c小鼠脾细胞CD4+T细胞混合培养3天,RT-PCR检测表明,T-DCs组诱导后CD4+T细胞Foxp3mRNA表达明显高于正常DCs、LPS-DC及Si-DC组。而Si-DC及LPS-DC刺激的CD4+T细胞Foxp3mRNA的表达明显降低(P0.05)。流式细胞仪检测表明,正常DCs、T-DCs、Si-DC及mDCs诱导的CD4+CD25+Treg细胞比例分别为(5.19±1.2)%、(28.29±2.36)%、(1.06±0.55) %,(2.01±0.66) %,以T-DC组诱导的Treg细胞比例明显增高(P0.01)。PI检测正常DCs、T-DCs、mDCs及Si-DC组诱导48h后CD4+T细胞的的凋亡率分别为(8.3±0.7)%、(21.56±2.32)%、(2.5±0.8)%、(1.9±0.7)%。3H检测异基因混合淋巴细胞增殖反应细胞刺激的增殖效应,且呈剂量依赖性。 结论诱导Treg细胞生成、促进异基因淋巴细胞凋亡是PIR-B介导性DCs耐受的分子机制,为临床应用耐受性DCs诱导免疫耐受提供理论依据,也为Treg的诱导提供新的途径。 第三部分CD8+CD28-T细胞对小鼠树突状细胞上配对免疫球蛋白样受体A和B表达的影响及与耐受的关系 目的诱导宿主产生供者主要组织相容性抗原的特异性耐受是临床骨髓移植的最终目标,CD8 + CD28-T(Ts)细胞是具有免疫抑制作用的调节性T细胞亚群之一,体外诱导异基因抗原特异性Ts生成,以研究Ts细胞与小鼠树突状细胞(DCs)上配对免疫球蛋白样受体A和B表达的关系,探讨其诱导免疫耐受的分子机制及特点,为临床抗原特异性免疫治疗的诱导提供理论基础。 方法体外诱导Ⅰ类主要组织相容性抗原(H-2b)抗原特异性Ts细胞群,以C57BL/6小鼠(H-2b)骨髓来源的树突状细胞系DC2.4细胞为刺激细胞,同BALB/c小鼠(H-2d)脾淋巴细胞混合培养,连续两次,每次培养7天,第10天于培养体系中加入IL-2(10u/ml),第14天结束培养。以生物素标记的CD28、CD8标记上述细胞群,以链亲和素标记的免疫磁珠分两步分选Ts细胞,首先负选CD8+T细胞,再正选CD28+T细胞,阴选细胞悬液为Ts细胞群。Ts同C57BL/6小鼠DC2.4(H-2b)细胞混合培养48h,RT-PCR检测DCs细胞PIR-A、PIR-BmRNA的表达,Westernblot检测DCs细胞PIR-A、B的表达。3H标记检测混合淋巴细胞增殖反应(MLR),体外诱导培养KM鼠骨髓来源的树突状细胞,分别以DC2.4(H-2b)及第三者主要组织相容性抗原无关的KM供鼠DCs细胞为刺激细胞,以BALB/c小鼠来源的脾CD4+T淋巴细胞为反应细胞,加入Ts细胞,以CPM检测异基因淋巴细胞增殖反应能力。 结果体外以C57BL/6小鼠DCs诱导并在体外应用免疫磁珠分选的CD8+、CD8+CD28-Ts 90%,以BALB/c小鼠Ts细胞(H-2d)与异基因C57BL/6小鼠DCs细胞(H-2b)混合培养48h后,RT-PCR及Western blot检测PIR-BmRNA及蛋白表达上调、PIR-A的mRNA及蛋白表达则下调。3H掺入标记检测显示,DC2.4细胞及KM鼠DCs细胞均可刺激BALB/c小鼠(H-2d)脾CD4+T淋巴细胞增殖,加入Ts细胞后,可以明显抑制DC2.4细胞刺激的BALB/c小鼠脾CD4+T淋巴细胞(H-2d)的增殖,而并不抑制KM鼠DCs细胞刺激的CD4+T脾淋巴细胞(H-2k)的增殖反应。 结论体外诱导的Ts呈现Ⅰ类主要组织相容性抗原特异性抑制异基因反应性淋巴细胞的增殖。其机制与Ts细胞上调PIR-B mRNA、下调PIR-A mRNA的表达有关。升高供体移植物中抗原特异性Ts细胞比例或受体树突状细胞表面免疫抑制性受体的表达可能成为诱导受者产生针对供体抗原特异性免疫耐受的有效途径。
[Abstract]:Graft-versus-host disease (GVHD) is still one of the major obstacles in the treatment of malignant diseases of the blood system in clinical bone marrow transplantation. Abnormal activation of allogeneic lymphocytes in donors is an important factor in the development of GVHD. It is the most important factor to induce the recipient to produce MHC specific tolerance against donor allogeneic major histocompatibility (MHC), which is the most important factor to prevent GVHD and to preserve GVL Paired immunoglobin-like receptor (PIR) is an immunosuppressive receptor that is mainly expressed in mouse dendritic cells (DCs), including immunosuppressive receptor (PIR-B) and immune activation receptor (PIR-A). The ligand of the ligand are MHC- I, the level of the binding of two to its ligand. It is one of the important molecular markers to determine the degree of immune activation of DCs. Because DC is not only the key to regulate the activation of T cells in the body, it is also an ideal target cell for inducing immune tolerance. It can directly or indirectly induce the generation of donor antigen specific regulatory T cells and form the order of T cells in the body to inhibit T lymphatic fining of the allogenic T. Therefore, the study of the expression of PIR in dendritic cells, the relationship of T cell order and the relationship with immune tolerance may provide a new way for inducing MHC specific tolerance and the separation of GVHD and GVL.
The first part is the expression of immunoglobulin like receptor on mouse dendritic cells and its relationship with immune tolerance.
Objective the paired immunoglobulin like receptor A, B (paired immunoglobulin-like receptor A, B, PIR-A, PIR-B) belong to the members of the mouse immunoglobulin superfamily. The relationship between the expression of paired immunoglobulin like receptor PIR-A, B on mice DCs and the changes in the surface costimulatory molecules, and the relationship between the immune tolerance and the immune tolerance are explored to explore the induction of tolerance. An effective way of being subjected to sexual DCs.
Methods the DC DC2.4 cells from C57BL/6 mice were used as the research object, and the recombinant human interleukin -10 (recombinant mouse interleukin-10, rmIL-10) and recombinant human transforming growth factor beta 1 (recombinant human transforming growth) were induced. 48h is a mature DC2.4 cell (LPS-DC), and the specific PIR-B small RNA interference fragment (small interfering RNA, siRNA) is synthesized in vitro (small interfering RNA, siRNA), and Lip2000 transfected to DC2.4 cells (Si-DC). The changes in the surface CO stimulatory molecules CD80, CD86, MHC- II and PIR-A were detected after LPS stimulation of Si-DCs, and the relative expression of cDNA transcript of each target gene was expressed with 2- Delta Delta Ct. The DCs cells in all of the above-mentioned groups were stimulated cells and the spleen lymphocytes of heterologous BALB/c mice were used as reactive cells, and the lymphatic allograft was detected by 3H-TdR labeling method. Cell proliferation ability (MLR), ELISA method was used to measure the secretion level of IFN- IFN- in supernatant.
Results FCM detected PIR-A on DC2.4 cells and the positive rate of PIR expression in the common extracellular domain of PIR-B was (28.65 + 8.12)%, IL-10, TGF- beta 1 and LPS induced PIR expression up up (P0.05), respectively (54.21 + 6.34)%, (58.78 + 4.70)%, (48.24 + 6.75)%, but IL-10, TGF- beta 1 and each group showed no significant difference. The expression of mRNA and protein in PIR-B was increased after DC2.4 cells were induced by IL-10 and TGF- beta 1, but the expression of PIR-A decreased, while LPS stimulation was the opposite, the mRNA and protein expression of PIR-A were increased and the expression of PIR-B decreased. The transfection rate of SiRNA positive control group was 93.12%. 86, the expression of MHC- II and PIR-A is higher than that of the normal DCs group.LPS-DCs group CD80, CD86, MHC- II and PIR-A's 2- Delta Delta Ct are 5.02 + 1.09,4.69 + + + 2.13 respectively. P0.05. The mixed lymphocyte reaction showed that normal DC2.4 cells could stimulate the reaction of allogeneic lymphocytes. The MLR in T-DC group induced by IL-10, TGF beta 1 was obviously inhibited (P0.05), and the level of IFN- gamma in MLR supernatant was also decreased (P0.05).LPS-DC and Si-DCs group increased significantly.
Conclusion up regulation of immunosuppressive receptor PIR-B and down-regulation of activated receptor PIR-A are the universal phenotypic characteristics and molecular biological mechanism of DCs tolerance in mice. The expression of silent PIR-B can make PIR-A and CD80, CD86, MHC-- II and PIR-A overexpressed. The mechanism of DCs activation, PIR-A and PIR-B are the new targets of mouse dendritic cell tolerance.
The second part of the highly expressed immunoglobulin like receptor B tolerant dendritic cells can induce the formation of CD4+CD25+ regulatory T cells.
Objective to study the relationship between the expression of paired immunoglobulin like receptor B and the production of regulatory T cells in dendritic cells, and to explore the detailed mechanism of tolerance induced tolerance of DCs, and to provide experimental basis for inducing tolerance DCs and regulatory T cells (Treg) in vivo.
The CD4+T splenic lymphocyte of BALB/c mice was selected by immunomagnetic beads. The DC2.4 cells derived from C57BL/6 mice were combined with rmIL-10 (50ng/ml) and rhTGF- beta 1 (50ng/ml) to induce the tolerance DCs (T-DC). The CD4+T splenic lymphocyte of /c mice was mixed with 48h, and the expression of Foxp3mRNA was detected by Treg generation.RT-PCR. The proportion of CD4+CD25+Treg cells was detected by flow cytometry. PI was used to detect the apoptosis of CD4+T cells. The CD4+CD25+Treg of magnetic beads and CD4+T cells were added to the MLR system according to the different specific cases. Stimulation of the proliferation ability of syngeneic lymphocytes.
Results the purity of CD4+T and CD4+CD25+T lymphocytes in the spleen of BALB/c mice was 95%. The cells of normal DCs, T-DCs, Si-DC and mDCs were mixed with the CD4+T cells of the spleen cells of BALB/c mice for 3 days. The RT-PCR detection showed that the expression of CD4+T cells was obviously higher than that of the normal group after the T-DCs group. The expression of Foxp3mRNA was significantly reduced (P0.05). Flow cytometry showed that the proportion of normal DCs, T-DCs, Si-DC and mDCs induced CD4+CD25+Treg cells was (5.19 + 1.2)%, (28.29 + 2.36)%, (1.06 + 0.55)%, (2.01 + 0.66)%, and the proportion of Treg cells induced by T-DC group was significantly higher (P0.01).PI detection of normal DCs. The apoptosis rate of CD4+T cells was (8.3 + 0.7)%, (21.56 + 2.32)%, (2.5 + 0.8)%, (1.9 + 0.7)%.3H, respectively, and (1.9 + 0.7)%.3H to detect the proliferation effect of allogeneic mixed lymphocyte proliferation reaction, and it was dose-dependent.
Conclusion induction of Treg cell production and promoting the apoptosis of allogeneic lymphocytes is the molecular mechanism of PIR-B mediated DCs tolerance, providing a theoretical basis for the clinical application of tolerance DCs to induce immune tolerance, and also provides a new way for the induction of Treg.
The third part is the effect of CD8+CD28-T cells on the expression of paired immunoglobulin like receptors A and B in mouse dendritic cells and their relationship with tolerance.
CD8 + CD28-T (Ts) cells are one of the regulatory T cell subsets with immunosuppressive effects, and the specific Ts generation of allogenic antigen in vitro can be induced in vitro, in order to study the paired immunity of Ts cells and mouse dendritic cells (DCs). The relationship between the expression of globulin like receptor A and B and the molecular mechanism and characteristics of its induced immune tolerance provide a theoretical basis for the induction of clinical antigen specific immunotherapy.
Methods the primary histocompatibility antigen (H-2b) antigen specific Ts cell group was induced in vitro. The dendritic cell line DC2.4 cells derived from C57BL/6 mice (H-2b) were used as stimulating cells and mixed culture with BALB/c mice (H-2d) splenic lymphocytes for two consecutive times, each time was 7 days, and IL-2 (10u/ml) was added to the culture system for tenth days and fourteenth days. At the end of the culture, CD28, CD8 labeled with biotin, were labeled with the above cell group, and the Ts cells were divided into two steps with the immunomagnetic beads labeled by streptavidin. First, the CD8+T cells were selected, and then the CD28+T cells were selected, the Ts cell group.Ts was mixed with the DC2.4 (H-2b) cells of the C57BL/6 mice, and the RT-PCR detected the expression of the cells. Westernblot detection of DCs cells PIR-A, B expression.3H markers to detect mixed lymphocyte proliferation response (MLR), and in vitro induction and culture of dendritic cells from the bone marrow of KM mice. DC2.4 (H-2b) and KM of the three main histocompatibility antigen independent KM mouse DCs cells are prickly cells. Ts cells were added into the cells, and the ability of allogeneic lymphocyte proliferation was detected by CPM.
Results in vitro, CD8+, CD8+CD28-Ts 90%, was induced by DCs in C57BL/6 mice and used in vitro by immunomagnetic beads. BALB/c mice Ts cells (H-2d) were mixed with DCs cells (H-2b) in C57BL/6 mice. The results showed that DC2.4 cells and KM mouse DCs cells stimulated the proliferation of CD4+T lymphocytes in the spleen of BALB/c mice (H-2d). After adding Ts cells, the proliferation of the spleen CD4+T lymphocyte (H-2d) of BALB/c mice stimulated by DC2.4 cells was obviously inhibited, but the proliferation reaction of spleen lymphocyte stimulated by KM mouse cells was not inhibited.
Conclusion Ts in vitro induced the proliferation of the main histocompatibility antigen specific inhibition of allogeneic reactive lymphocytes. The mechanism is related to the up regulation of PIR-B mRNA, down regulation of the expression of PIR-A mRNA, the increase of the proportion of antigen specific Ts cells in donor grafts or the expression of immunosuppressive receptors on the surface of the receptor tree process cells. It may be an effective way to induce recipient to produce donor specific antigen tolerance.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R392
本文编号：2128714
[Abstract]:Graft-versus-host disease (GVHD) is still one of the major obstacles in the treatment of malignant diseases of the blood system in clinical bone marrow transplantation. Abnormal activation of allogeneic lymphocytes in donors is an important factor in the development of GVHD. It is the most important factor to induce the recipient to produce MHC specific tolerance against donor allogeneic major histocompatibility (MHC), which is the most important factor to prevent GVHD and to preserve GVL Paired immunoglobin-like receptor (PIR) is an immunosuppressive receptor that is mainly expressed in mouse dendritic cells (DCs), including immunosuppressive receptor (PIR-B) and immune activation receptor (PIR-A). The ligand of the ligand are MHC- I, the level of the binding of two to its ligand. It is one of the important molecular markers to determine the degree of immune activation of DCs. Because DC is not only the key to regulate the activation of T cells in the body, it is also an ideal target cell for inducing immune tolerance. It can directly or indirectly induce the generation of donor antigen specific regulatory T cells and form the order of T cells in the body to inhibit T lymphatic fining of the allogenic T. Therefore, the study of the expression of PIR in dendritic cells, the relationship of T cell order and the relationship with immune tolerance may provide a new way for inducing MHC specific tolerance and the separation of GVHD and GVL.
The first part is the expression of immunoglobulin like receptor on mouse dendritic cells and its relationship with immune tolerance.
Objective the paired immunoglobulin like receptor A, B (paired immunoglobulin-like receptor A, B, PIR-A, PIR-B) belong to the members of the mouse immunoglobulin superfamily. The relationship between the expression of paired immunoglobulin like receptor PIR-A, B on mice DCs and the changes in the surface costimulatory molecules, and the relationship between the immune tolerance and the immune tolerance are explored to explore the induction of tolerance. An effective way of being subjected to sexual DCs.
Methods the DC DC2.4 cells from C57BL/6 mice were used as the research object, and the recombinant human interleukin -10 (recombinant mouse interleukin-10, rmIL-10) and recombinant human transforming growth factor beta 1 (recombinant human transforming growth) were induced. 48h is a mature DC2.4 cell (LPS-DC), and the specific PIR-B small RNA interference fragment (small interfering RNA, siRNA) is synthesized in vitro (small interfering RNA, siRNA), and Lip2000 transfected to DC2.4 cells (Si-DC). The changes in the surface CO stimulatory molecules CD80, CD86, MHC- II and PIR-A were detected after LPS stimulation of Si-DCs, and the relative expression of cDNA transcript of each target gene was expressed with 2- Delta Delta Ct. The DCs cells in all of the above-mentioned groups were stimulated cells and the spleen lymphocytes of heterologous BALB/c mice were used as reactive cells, and the lymphatic allograft was detected by 3H-TdR labeling method. Cell proliferation ability (MLR), ELISA method was used to measure the secretion level of IFN- IFN- in supernatant.
Results FCM detected PIR-A on DC2.4 cells and the positive rate of PIR expression in the common extracellular domain of PIR-B was (28.65 + 8.12)%, IL-10, TGF- beta 1 and LPS induced PIR expression up up (P0.05), respectively (54.21 + 6.34)%, (58.78 + 4.70)%, (48.24 + 6.75)%, but IL-10, TGF- beta 1 and each group showed no significant difference. The expression of mRNA and protein in PIR-B was increased after DC2.4 cells were induced by IL-10 and TGF- beta 1, but the expression of PIR-A decreased, while LPS stimulation was the opposite, the mRNA and protein expression of PIR-A were increased and the expression of PIR-B decreased. The transfection rate of SiRNA positive control group was 93.12%. 86, the expression of MHC- II and PIR-A is higher than that of the normal DCs group.LPS-DCs group CD80, CD86, MHC- II and PIR-A's 2- Delta Delta Ct are 5.02 + 1.09,4.69 + + + 2.13 respectively. P0.05. The mixed lymphocyte reaction showed that normal DC2.4 cells could stimulate the reaction of allogeneic lymphocytes. The MLR in T-DC group induced by IL-10, TGF beta 1 was obviously inhibited (P0.05), and the level of IFN- gamma in MLR supernatant was also decreased (P0.05).LPS-DC and Si-DCs group increased significantly.
Conclusion up regulation of immunosuppressive receptor PIR-B and down-regulation of activated receptor PIR-A are the universal phenotypic characteristics and molecular biological mechanism of DCs tolerance in mice. The expression of silent PIR-B can make PIR-A and CD80, CD86, MHC-- II and PIR-A overexpressed. The mechanism of DCs activation, PIR-A and PIR-B are the new targets of mouse dendritic cell tolerance.
The second part of the highly expressed immunoglobulin like receptor B tolerant dendritic cells can induce the formation of CD4+CD25+ regulatory T cells.
Objective to study the relationship between the expression of paired immunoglobulin like receptor B and the production of regulatory T cells in dendritic cells, and to explore the detailed mechanism of tolerance induced tolerance of DCs, and to provide experimental basis for inducing tolerance DCs and regulatory T cells (Treg) in vivo.
The CD4+T splenic lymphocyte of BALB/c mice was selected by immunomagnetic beads. The DC2.4 cells derived from C57BL/6 mice were combined with rmIL-10 (50ng/ml) and rhTGF- beta 1 (50ng/ml) to induce the tolerance DCs (T-DC). The CD4+T splenic lymphocyte of /c mice was mixed with 48h, and the expression of Foxp3mRNA was detected by Treg generation.RT-PCR. The proportion of CD4+CD25+Treg cells was detected by flow cytometry. PI was used to detect the apoptosis of CD4+T cells. The CD4+CD25+Treg of magnetic beads and CD4+T cells were added to the MLR system according to the different specific cases. Stimulation of the proliferation ability of syngeneic lymphocytes.
Results the purity of CD4+T and CD4+CD25+T lymphocytes in the spleen of BALB/c mice was 95%. The cells of normal DCs, T-DCs, Si-DC and mDCs were mixed with the CD4+T cells of the spleen cells of BALB/c mice for 3 days. The RT-PCR detection showed that the expression of CD4+T cells was obviously higher than that of the normal group after the T-DCs group. The expression of Foxp3mRNA was significantly reduced (P0.05). Flow cytometry showed that the proportion of normal DCs, T-DCs, Si-DC and mDCs induced CD4+CD25+Treg cells was (5.19 + 1.2)%, (28.29 + 2.36)%, (1.06 + 0.55)%, (2.01 + 0.66)%, and the proportion of Treg cells induced by T-DC group was significantly higher (P0.01).PI detection of normal DCs. The apoptosis rate of CD4+T cells was (8.3 + 0.7)%, (21.56 + 2.32)%, (2.5 + 0.8)%, (1.9 + 0.7)%.3H, respectively, and (1.9 + 0.7)%.3H to detect the proliferation effect of allogeneic mixed lymphocyte proliferation reaction, and it was dose-dependent.
Conclusion induction of Treg cell production and promoting the apoptosis of allogeneic lymphocytes is the molecular mechanism of PIR-B mediated DCs tolerance, providing a theoretical basis for the clinical application of tolerance DCs to induce immune tolerance, and also provides a new way for the induction of Treg.
The third part is the effect of CD8+CD28-T cells on the expression of paired immunoglobulin like receptors A and B in mouse dendritic cells and their relationship with tolerance.
CD8 + CD28-T (Ts) cells are one of the regulatory T cell subsets with immunosuppressive effects, and the specific Ts generation of allogenic antigen in vitro can be induced in vitro, in order to study the paired immunity of Ts cells and mouse dendritic cells (DCs). The relationship between the expression of globulin like receptor A and B and the molecular mechanism and characteristics of its induced immune tolerance provide a theoretical basis for the induction of clinical antigen specific immunotherapy.
Methods the primary histocompatibility antigen (H-2b) antigen specific Ts cell group was induced in vitro. The dendritic cell line DC2.4 cells derived from C57BL/6 mice (H-2b) were used as stimulating cells and mixed culture with BALB/c mice (H-2d) splenic lymphocytes for two consecutive times, each time was 7 days, and IL-2 (10u/ml) was added to the culture system for tenth days and fourteenth days. At the end of the culture, CD28, CD8 labeled with biotin, were labeled with the above cell group, and the Ts cells were divided into two steps with the immunomagnetic beads labeled by streptavidin. First, the CD8+T cells were selected, and then the CD28+T cells were selected, the Ts cell group.Ts was mixed with the DC2.4 (H-2b) cells of the C57BL/6 mice, and the RT-PCR detected the expression of the cells. Westernblot detection of DCs cells PIR-A, B expression.3H markers to detect mixed lymphocyte proliferation response (MLR), and in vitro induction and culture of dendritic cells from the bone marrow of KM mice. DC2.4 (H-2b) and KM of the three main histocompatibility antigen independent KM mouse DCs cells are prickly cells. Ts cells were added into the cells, and the ability of allogeneic lymphocyte proliferation was detected by CPM.
Results in vitro, CD8+, CD8+CD28-Ts 90%, was induced by DCs in C57BL/6 mice and used in vitro by immunomagnetic beads. BALB/c mice Ts cells (H-2d) were mixed with DCs cells (H-2b) in C57BL/6 mice. The results showed that DC2.4 cells and KM mouse DCs cells stimulated the proliferation of CD4+T lymphocytes in the spleen of BALB/c mice (H-2d). After adding Ts cells, the proliferation of the spleen CD4+T lymphocyte (H-2d) of BALB/c mice stimulated by DC2.4 cells was obviously inhibited, but the proliferation reaction of spleen lymphocyte stimulated by KM mouse cells was not inhibited.
Conclusion Ts in vitro induced the proliferation of the main histocompatibility antigen specific inhibition of allogeneic reactive lymphocytes. The mechanism is related to the up regulation of PIR-B mRNA, down regulation of the expression of PIR-A mRNA, the increase of the proportion of antigen specific Ts cells in donor grafts or the expression of immunosuppressive receptors on the surface of the receptor tree process cells. It may be an effective way to induce recipient to produce donor specific antigen tolerance.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R392
【共引文献】
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